BASOLATERAL SECRETION OF AMYLOID PRECURSOR PROTEIN IN MADIN-DARBY CANINE KIDNEY-CELLS IS DISTURBED BY ALTERATIONS OF INTRACELLULAR PH AND BY INTRODUCING A MUTATION ASSOCIATED WITH FAMILIAL ALZHEIMERS-DISEASE

The analysis of potential sorting signals in amyloid precursor protein (APP) by site-directed mutagenesis and the disturbance of metabolic p athways by drugs is used here to define the parameters that determine polarized secretion of APP in Madin-Darby canine kidney cells. Endogen ously produced APP751/770 and APP695 produced from transfected constru cts are secreted almost exclusively into the basolateral compartment. The sorting mechanism is highly dependent on intracellular pH as demon strated by its sensitivity to primary amines and inhibitors of the aci difying vacuolar proton ATPase. The role of potential basolateral sort ing signals in the cytoplasmic, transmembrane, and beta A4 amyloid reg ion of APP was investigated. Neither deletion of the endocytosis and p utative basolateral sorting signal GY.NPTY nor complete deletion of th e cytoplasmic domain causes apical secretion of soluble APP. Further d eletion of the transmembrane domain and of the beta A4 amyloid region confirmed that the major basolateral sorting determinant resides in th e extracellular domain of APP. Increased beta-secretase cleavage of AP P after introduction of the ''swedish'' double mutation causes apical missorting of about 20% of beta-secretase-cleaved APP. The data underl ine the complexity of processing and sorting APP in polarized cells an d suggest a possible problem of protein sorting in Alzheimer's Disease .